US 2023/0416846 A1 · DIGITAL CRISPR-BASED METHOD FOR THE RAPID DETECTION AND ABSOLUTE QUANTIFICATION OF NUCLEIC ACIDS

Abstract

The present invention relates to a digital CRISPR-based method for detecting and quantitating target nucleic acids in a sample comprising: forming a mixture comprising sample nucleic acids; isothermal amplification reaction reagents for amplifying one or more target nucleic acid sequences; partitioning the mixture into a plurality of compartments; incubating the partitioned mixture at a temperature for isothermal amplification and Cas effector cleavage of an amplified DNA strand, detecting a signal from cleavage of the non-target sequence, thereby detecting the one or more target sequences in the sample, and determining the copy number of the target nucleic acid based on a Poisson distribution of the proportion of positive-to-negative compartments. The invention also relates to a method for detecting presence and/or of a disease in a subject, and a kit to quantitate nucleic acids in a sample.

Claims27

Dependent claims

A method for detecting and quantitating target nucleic acids in a sample comprising:

a) forming a mixture comprising sample nucleic acids;
isothermal amplification reaction reagents for amplifying one or more target nucleic acid sequences;
a Cas12a, Cas12b, Cas13b or Cas14 effector, or a derivative thereof;
at least one guide polynucleotide comprising a DNA-targeting sequence, and designed to form a complex with the Cas effector;
and
a nucleic acids-based masking construct comprising a non-target sequence,
b) partitioning the mixture into a plurality of compartments;
c) incubating the partitioned mixture at a temperature for isothermal amplification and Cas effector cleavage of an amplified DNA strand,
wherein the Cas effector exhibits collateral nuclease activity and cleaves the non-target sequence of the nucleic acids-based masking construct once activated by the target sequences;
and
d) detecting a signal from cleavage of the non-target sequence, thereby detecting the one or more target sequences in the sample, and
e) determining the copy number of the target nucleic acid based on a Poisson distribution of the proportion of positive-to-negative compartments.

9 more elements

A method for detecting the presence and/or severity of a disease in a subject, comprising the steps of:

a) forming a mixture comprising a sample comprising nucleic acids from the subject;
isothermal amplification reaction reagents for amplifying one or more target disease nucleic acid sequences;
a Cas12a, Cas12b, Cas13b or Cas14 effector, or a variant thereof;
at least one guide polynucleotide comprising a DNA-targeting sequence, and designed to form a complex with the Cas effector;
and
a nucleic acids-based masking construct comprising a non-target sequence,
b) partitioning the mixture into compartments;
c) incubating the partitioned mixture at a temperature for isothermal amplification and Cas effector cleavage of an amplified DNA strand,
wherein the Cas effector exhibits collateral nuclease activity and cleaves the non-target sequence of the nucleic acids-based masking construct once activated by the target sequences;
d) detecting a signal from cleavage of the non-target sequence, thereby detecting the one or more target sequences in the sample;
e) determining the copy number of the target nucleic acid based on a Poisson distribution of the proportion of positive-to-negative compartments and comparing the number to a control value;
wherein positive compartments indicate the presence of disease in said subject, and wherein the copy number of the target nucleic acid indicates the severity of the disease in said subject.

9 more elements

Dependencies are inferred from the claim text.

Digital crispr-based method for the rapid detection and absolute quantification of nucleic acids

Life of this patent

Digital crispr-based method for the rapid detection and absolute quantification of nucleic acids

Life of this patent

Abstract

Claims27